Modifications by light of synaptic density in the inner plexiform layer of the toad, Xenopus laevis

ESPEKIMENTAI.

XELXOLOGY

Modifications Plexiform GAIL

55.

133-151

(1977)

by Light of Synaptic Layer of the Toad, SUSAN

TUC.I~ER

AND

JOE (;.

Density in the Xenopus laevis HOLLYFIELD

Inner

1

Retinal differentiation was analyzed quantitatively in light- and dark-reared larvae of the toad Srrtop~rs larzris. Simple and serial conventional synapses and bipolar cell synaptic ribbons in the inner plexiform layer were measured from electron micrographs and their volume densities calculated. Photoreceptor synaptic ribbons were measured from electron micrographs. Inner nuclear layer cell densities were determined and thicknesses of various retinal layers measured from light micrographs of thick-sectioned material. In light-reared larvae, simple synapse density is stable through Stage 52 of development (‘f = 299 synapses/1000 +n?‘) _ At Stage 53 through Stage Meta’ (5 months postmetamorphic), synaptic density is low ( < 200 synapses/1000 pm’). Xt Stage hIeta++ (8 months postmetamorphic) it increases to the adult level (250 synapses/1000 pm”). In dark-reared larvae, simple synapse density is greater than in light-reared larvae at all stages examined (.T = 378/1000 pm3). \2’hen dark-reared larvae are exposed to cyclic light at Stages 46 and 48, synaptic density returns to light-reared larval values by Stages 52 and 53, respectively. Dark-reared larvae show partial recovery when exposed to light at Stage 51 but do not respond to light exposure at Stage Meta+. Serial synaptic density does not change significantly during development in lightor darkreared larvae. Bipolar cell synaptic ribbon density is high in light-reared larvae through Stage 51. Ribbon density then decreases to a low level at Stage j2 which is maintained beyond metamorphosis. Dark-reared larvae exhibit the same pattern through Stage 51. By Stage 54. ribbon density is three times that in light-reared, Stage-54 larvae. In dark-reared Meta” larvae, ribhon density is nearly four times that in the corresponding light-reared larvae, In dark-reared larvae exposed to cyclic light at Stage 46, ribbon density returns 1 This

\vork \vas supported hy National Iiistitutes of Health, Research Grant EYand Training Grants EY-000?9-1-l and EY-07007-01, while GST was a Postdoctoral Fellow at Columbia University, College of Physicians and Surgeons, Department of Ophthalmology. JGH is the recipient of a Research Career Development Award l-KWEY-00023. Dr. Tucker’s present address is Bascom Palmer Eye Institute Cniversity of Miami School of Medicine. 1638 I\.\V. 10th ;\venue, Xliami, Florida, 33152.

OO6?4

Copyright

0

1977

by

Academic I’rrss, Inc.

134

TUCKER

AND

HOLLYFIELD

to normal, but at later stages (48, 51, and Meta+), dark-reared larvae do not respond to light during the time intervals allowed. The average lengths of rod cell outer segments and the thickness of the inner plexiform layer decrease significantly in dark-reared larvae.

INTRODUCTION Numerous studies detailing the effects of modified sensory input on the adult and developing nervous system have been reported (1, 15, 18). Neuronal tissues have been shown to undergo morphological and physiological responses to long- and short-term variations in sensory input in nearly every class of vertebrate. In studies dealing with the visual system an interplay between sensory input and the differentiation and/or maintenance of neuronal components has been suggested. Visual deprivation caused by eye removal or occlusion results in alterations in the optic tecta of lower vertebrates. In mammals, responses to visual deprivation have been reported in the lateral geniculate nucleus, in the superior colliculus, and in the visual cortex. Several studies indicate that if visual deprivation is to result in permanent impairment of higher visual centers, it must occur during a critical period of development (9, 14, 19, 22, 40). In the retina, visual deprivation can result in decreased thickness of the inner plexiform layer (29, 42). Altered enzyme activities and a loss of RNA have been observed in deprived retinae (23, 31). After even shortterm dark adaptation, synaptic ribbons shorten in the inner plexiform layer and in cone pedicles (41) . In this study we determined values for inner plexiform synaptic volume density in retinae from larval and young adult Xenoplts laevis reared either under a controlled light/dark cycle or in complete darkness. Because dark-rearing was found to elevate synaptic density, we assessed the permanence of this morphological response by determining the values for synaptic density in dark-reared animals after their introduction at various stages to of this study have been reported the light/dark regimen. Portions previously.2 MATERIALS

AND

METHODS

Spc&nens. Adult Xenopzrs laevis males and females were injected with gonadotrophic hormone (A.P.L., Ayerst) according to the method of Gurdon (16) to induce amplexus, and gamete maturation and release. At the yolk plug stage, Stage 10 [see (25) for staging criteria], batches of 100 embryos each were placed in separate aquaria, one exposed to diurnal light and the other in complete darkness. 2 TUCKER, G. S. 1975. Synaptogenesis in the IPL of light- and dark-reared Imis larvae. Assoc. Res. Vision Ophthalmol., Annu. Meeting, Sarasota, 1975. Abst.; Responses of inner plexiform synapses to dark-rearing in the the toad, Sruopus 1uevi.s. Neurosci. Abst. 1 : 98.

Xe~opus Florida, retina of

IXh‘ER

I’LESIFOR~I

SYNAPTIC

DENSITY

AKD

LI(;IIl

13.5

Reavi~g Cor~Gtiorts. Animals were reared in continuously aerated 10% Holtfreter’s solution at 20 * 1°C. Tadpoles were maintained in lo-gal (37.85-m”) aquaria and fed with suspensions of powdered nettles (16). At Stage 60, metamorphosing animals were transferred to individual finger bowls and hand-fed to satiation twice weekly with small fragments of beef liver. Control aquaria were covered with a glass plate wrapped in Parafilm. i\ rack of three 40-M’ tungsten light bulbs 100 cm above the aquarium surface provided the fight source, and a timer produced 8-h light: 16 h-darkness daily. The light intensity measured at the aquarium surface was 125 lux. Dark-reared animals were placed in an aquarium and kept in the dark. Photographic plates placed on top of the aquarium remained unexposed throughout the cxperimcnt as shown by photographic development. Dark-reared animals were fed under dim red light. Just prior to metamorphosis animals were transferred to individual finger bowls but remained in the dark as before. Recovery animals were reared in the dark to Stages 46. 48, 51, and Meta+ (5 months postmetamorphic). At each of these stages they were placed in individual finger bowls under the same lighting conditions as light-reared animals. They were killed, rcspcctively, at Stages 52, 53, 54, and Meta’+ (8 months postmetamorphic). Slrcfron ;llicrclscopy. Preparations for fixation of embryonic eyes were done under dim red light and adult eyes were cut up and fixed under room light. Eyes were fixed 90 min at 4°C in a mixture of 1.87% glutaraldchyde and 1% osmium tetroxide in 0.05 11 phosphate buffer (pH 7.4) with 0.05 at sodium chloride. The osmolarity of the fixative was 380 mos>1 as measured with a freezing osmometer. The tissue was rinsed momentarily in the cold in 0.05 JI phosphate buffer and then in sodium maleate buffer (pH 4.5) and stained cu bloc 1 11 in 2% uranyl acetate in maleate buffer. After dehydration at 4°C in graded ethanol and propylene oxide, the tissue was embedded in Epon. Eyes were oriented in the unpolymerized Epon with the sagittal plane parallel to the block face. Following polymerization, l-prt-thick sections were cut until the optic nerve was reached. The block was then trimmed so that a strip of dorsal retina containing a tab of optic nerve remained. In the adult this segment was selected prior to embedment. Thin sections were taken on a diamond knife and mounted on Parlodioncoated slot grids. The sections were stained with saturated uranyl acetate in 25% acetone-75% ethanol followed by lead citrate (30) and photographed in a Siemens Elmiskop 1A electron microscope. Figure 1 shows the area examined in our study. An area dorsal to the optic nerve was selected because in S. loc7G this region of the retina is the first to begin differentiating (37). At a distance of 60 Frn (or more) beyond the dorsal border of the optic nerve head, series of photographs were taken at X8000 to produce complete mosaics of the entire thickness of the inner plexiform layer (photographic enlargement to ~24,000). Survey photographs of rod and cone receptor bases, pigment epithelium, receptor cell outer segments, nuclear layers, and the ganglion cell layer were also taken. Ultrastructlryal Crifcrin for Synapse Idmtificafior~. The ultrastructure of synapses in the imler plexiform layer of the retina has been described previously (4-8, 13, 20, 21, ‘24, 35, 44). Using these descriptions we have set criteria for identification of inner plexiform synapses in X. larztis. Corrzle+ttio& synapses are characterized by presynaptic and postsynaptic membrane densities, a cluster of synaptic vesicles (at least three) on the presynaptic side, and broadening of the intermembrane space at the synapse site. Conventional synapses may be arranged in either silt@ or scvial COXIfiguration and may be axo-axonic (amacrine to amacrine), axo-dendritic (amacrine to ganglion cell dendrite), or axo-somatic (amacrine to ganglion cell body or inner nuclear layer cell body). In addition. a very few bipolar cell synapses lacking an

136

TUCKER

AND

HOLLYFIELD

80~ 601~ 4w 2OA

FIG. 1. Diagram of the larval eye of S~~D~~IIS lnrz~ir. Enlargement at the right shows the location and arrangement of the various retinal layers and the appearance of sections used in this study for light and electron microscopy. PE--pigmented epithelium; GCL-ganglion cell layer, INL-inner nuclear layer, IPL-inner plexiform layer, D-dorsal, V-ventral.

associated ribbon were classified as conventional in this study. Like amacrine cell synapses these were either axe-axonic or axo-dendritic but not alto-somatic. We did not examine serially sectioned material to determine whether or not an associated ribbon was present in sections adjacent to these conventional type bipolar synapses (33). Nor did we attempt in this study to provide an analysis of the relative occurrence of axe-axonic. axe-dendritic, or axo-somatic connections (36). Simple conventional synapses are presynaptic to a neurite which is not itself presynaptic to another neurite in the same field. Scriul conventional synapses are presynaptic to another neurite which is itself presynaptic to a third neurite, and so on (we have seen as many as four such synapses arranged interconnecting five separate nerve processes). Serial synaptic configurations of several types are seen, including amacrine to amacrine (Fig. 2: all serial synapses illustrated are of this type) ; amacrine to bipolar (Fig. 3) ; and reciprocal synapses in which each member is presynaptic to the other (Fig. 2, S, and S,). Whenever the term “conventional” is used, we are referring to simple and serial conventional synapses collectively. Unless otherwise indicated, each serial synapse was counted as a single synapse and was not included in the data .with simple conventional synapses. Bipolar cell synaptic ribbon configurations are characterized by presynaptic and postsynaptic membrane densities and a dense bar or ribbon of electron-opaque material in the presynaptic process, oriented in a variety of ways with respect to the membrane specialization and surrounded by a halo of synaptic vesicles. In our ma-

INNER

PLEXIFORM

SYXAPTIC

DENSITY

APiD

LIGHT

137

terial, two morphologically distinct types of bipolar cell terminals containing synaptic ribbons were seen in the inner plexiform layer. These we have termed “dense” and “clear” bipolars (Fig. 3-5). Dense bipolars contain dense cytoplasm, tightly packed synaptic vesicles, mitochondria, and, frequently, small irregularly shaped electrontransparent vesicles of undetermined significance. Compared to dense bipolar cell terminals, clear bipolars have fe\ver synaptic vesicles and few cellular organelles and a less dense cytoplasm. Q2tnrlfiftrfiort of Kesulfs. Mosaic areas were measured with a compensating polar planimeter, and the lengths of conventional synapse profiles and bipolar cell synaptic ribbons were measured with a vernier caliper. A thickness of 600 .4 was assumed fur sections reflecting grey to silver-grey interference colors. These data were then used to compute a value, the number of synapses or ribbons per 1000 pm’ of inner plexiform layer [see (7) for method]. Extreme curvature of conventional synapses, resembling “spine” synapses (20), was indicated on data sheets in order to quantify the incidence of this characteristic. In electrically stimulated superior cervical ganglia, stimulation results in increased zones of contact between presynaptic and postsymaptic neuritcs (27, 28). The volume density of nuclei in the inner nuclear layer was calculated using measurements of nuclear diameter and planimetry measurements of area and applying Dubin’s formulation (7) to I-pm-thick sections. We calculated the average total length of synaptically specialized membrane per unit area of inner plexiform layer present in our specimens. This was done by adding together all conventional synapse measurements for a particular set of mosaics and correcting their size to the length of synapses per 1000 pm’ of inner plexiform layer. Data from all measurements were compared using Student’s f test to determine the significance of differences in these parameters at different stages of development and under different experimental conditions. Confidence intervals are derived from the standard error of the mean of measurements of computed volume densities from all light-reared, dark-reared, or recovery larvae specimens at each stage (for example, five mosaics at Stage 43 were compared from one light-reared larva). The average area per mosaic was 870 pm”. Table 1 indicates the number of replicates and specimens for each stage and treatment. Additional specimens were included for the analyses of the inner nuclear layer, inner plexiform thickness, and rod cell outer segment lengths. In addition to our evaluation of synapse density in the inner plexiform layer of lightand dark-reared larvae, measurements were made of inner plexiform layer thickness, photoreceptor cell ribbons, rod cell outer segment length, and thickness of the inner nuclear layer. Sections \vere cut in the vertical plane. The block face ~vas oriented such that the optic nerve head and a tab of epidermal tissue connecting the midpoint of the anterior lens with the cornea could be seen in the same section (Fig. 1 ) . Only those rod cell outer segments which were located at the posterior pole of the eye ant] were vlslble through their entire length were measured. These same thick sectiolls \vere used for measurements of other retinal layers reported upon here.

OBSER\-XTIOSS

Tlzicbzess. At selected stages of developwent, the thickness of the inner plexiform layer was measured from light micrographs of l-pm Epon sections. These data are given in Fig. 6. There were no significant differences in layer thickness in light- and dark-reared larvae during larval develop-

138

TUCKER

AND

HOLLYFIELD

Z-F-P

000

Lc-4

000

13r3

3-4

tT4.w

000

000

000

000

000

2 2-z -o- -

2

INNER

PLEXIFORM

SYNAPTIC

DENSITY

AND

LIGHT

139

FIG. 2. Serial conventional synapses in the inner plexiform layer of .Y. Loomis ( S, and Sl:, through S,, arrows ). -b\ reciprocal synapse is she\\ n ( S., and S,) Calibration bar is the same for Fig. 2 through 5. FIG. 3. Serial synaptic arrangements in which amacrinc cell neurites are prr(D) bipolar terminal. GCL-ganglion cell. synaptic (5% and S,) to a “dense” FIG. 4. i\ dense bipolar cell synaptic terminal in which an electron-opaque cytoplasm packed with synaptic vesicles, mitochondria, and electron transparent vesicles is shown. FIG. 5. A clear bipolar cell profile (CL, arrow) having few cellular organelles and electron-transparent cytoplasm is shown. The synaptic ribbon (arrowheads) is not located immediately adjacent to the membrane density.

140

TUCKER

AND

IIOLLYFIELD

Developmental stage 6. The thickness of the inner plexiform layer (y-axis) is plotted against developmental stage (x-axis>. In Figs. 6-8, open circles (0 . * . 0) indicate data for light-reared larvae (LRL) ; closed circles ( 0-O) indicate values for darkreared larvae (DRL). Stages are arranged chronologically but not proportionately Confidence intervals are derived according to time in this and succeeding figures. from the standard error of the mean of density values calculated from several mosaics at each point. FIG.

merit. The thickness of the inner nuclear layer in light-reared larvae was significantly greater than in dark-reared larvae at Stage Meta++ (P < 0.05). Sil+tplc Conventional Synapses. In evaluating the density of simple synapses proximal to and at varying distances (0 to 500 pm) from the optic nerve head, we found that simple synapse density was constant beyond 60 to 100 pm dorsal to it. At distances less than 60 pm, calculated volume density values were quite variable and extremely high. Variability in dark-reared larvae was even greater than that observed in light-reared animals. Yazulla (44) found differences in synaptic density in different regions of pigeon retina. Therefore, in this study, data from the dorsal retina were collected only at 60 pm from the optic nerve head and beyond (see Fig. 1). The densities of simple synapses in the inner plexiform layer of lightreared,

dark-reared,

and

recovery

larvae

at

different

stages

of

larval

and

postmetamorphic development are presented in Table 2. At premetamorphic stages there are significantly more simple synapses in dark- than in lightreared larvae, whereas in postmetamorphic animals this difference is not significant (Fig. 7). The density of simple synapses in recovery larvae also varies from that in light-reared animals. Table 3 gives the schedule of dark- versus lightrearing for each recovery animal studied and indicates the length of the recovery period in days. Simple synapse density data for the recovery larvae are given in Fig. 7. In

recovery

larva

R1

(dark-reared

to

Stage

46,

recovery

from

Stage

46

IKiXER

PLEXIFORM

SYNAPTIC

DENSITY

TABLE Inner

Plexiform

Stage

43 47 48 50 51 52 53 54 Meta+ Meta++ Adult

Layer

Synapse

141

LIGHT

2

Density

Light-reared

AND

1.alues

(Number/1000

Dark-reared

Recover?

Simple

Ribbon

Serial

Simple

267 358 277 296 302 319 198 169 173

147 123 144 128 100 48 71 47 42

27 32 21 24 33 20 29 21 24

468

378

124

.Z2

249 2.54

31 106

41 38

343 -

140

30

Kibbon

Serial

Simple

156 143 128 116 51

36 41

-

35

-

54 30

476 4 13

420 399

prna)

Ribbon

Serial

-

-

181 299 243

-

43 91 87

-

9 18 20 -

411 -

115

28

-

-

to Stage 52). the number of simple synapses is less than in light-reared larvae at Stage 52. In Rz (dark-reared to Stage 48, recovery Stages 48 to 53), the value is elevated and is not significantly different from light-reared Stage 53 but is significantly less than dark-reared Stage 45. In Ra (darkreared to stage 51, recovery Stages 51 to 54)) there are significantly more simple synapses than in light-reared Stage 54 and significantly fewer than

I

*

.

.

.

.

43

47

48

50

51

Developmental FIG.

y-axis, through

52

o......o

LR L

.-.

DRL

.

.

53

54

1

.

m+ m*+

.

adult

stage

7. Graph of simple conventional synapse densities. S-.ixis, number of synapses per 1000 ,&’ of inner plexiform layer. R,-recovery group designation (see Table 3).

developmental stage : In Figs. 7 and 8, RI

142

TUCKER

AND

HOLLYFIELD

TABLE Recovery Recovery group designation

RI RP R, R4

-

0 Significant

3 Schedule

Stage

of development in dark (days in dark)

Stage at recovery (age in days)

46 (6) 48 (7) 51 (10) Meta+ (8 months) recovery

Length of recovery period (days)

52
20 40 40 100

noted.

in dark-reared Stage 54. In addition, Ra has significantly fewer simple synapses than does dark-reared Stage 5 1. R4 (5 months postmetamorphic dark-reared, 3-month recovery period) has significantly more simple synapses than light-reared Meta++. Simple and serial conventional synapses are larger in light-reared than in dark-reared and recovery larvae (Table 4). Synapses in the former are significantly larger than those in the latter only at Stage 54 (P < 0.001) and larger than those of recovery larvae at Meta” (P < 0.05). There are no significant differences in size between simple and serial conventional synapses at any stage in any specimen. 1L’hen one calculates the amount of synaptically specialized membrane per 1000 +19 of inner plexiform layer one finds that for the most part there is no difference between light-reared, dark-reared, and recovery larvae (Table 4). The incidence of curved conventional [spine : (20) 1 synapses does not change from stage to stage and is the same in all specimens (7/1000 pm”). TABLE Conventional

stage

Light-reared pm/syna),se (9)

43 47 48 50 51 52 5.3 54 Meta+ Meta+’ Adult

4

Synapse

0.3224 0.3400 0.353’3 0.3176 0.3658 0.3579 0.3555 0.3730 0.4266 0.4497 0..3776

Lengths

Dark-reared

/dn/1000

(.Y /A) 26.8 40.3 32.2 30.9 36 2 35 3 23.1 23.1 30.6 38.4 36.3

/An12

*m/synapse

Recovery

pm/l000

pm2

jm/synavse

rm/1000pm2

(S)

(2 IA )

(9)

(9/A)

0.3050 0.2546 0.3181 0.2981 0.3253 0.3231 0.3704 -

45.1 32. I 35.4 37.0 38.4

-

22.6 31.0 47.0

23.1 44.1

0.3727 0.3278 0.3505

IiXh-ER

PLEXIFORM

SYSAPTIC

DENSITY

o......

.-.

Developmental

8. Graph of bipolar same as in Fig. 7. FIG.

0

i’.?;D

LICIIT

143

L R L

DRL

stage

cell synaptic ribbon densities. S-Axis

and y-axis are the

Sy~zaptic Ribbons. The density of synaptic ribbons in the inner plexiform layer of al! specimens,was calculated and compared at different stages of development. In light- and dark-reared larvae the value is elevated at early stages of development (Fig. 8, Table 2). By Stage 52 it decreasesin lightreared larvae and remains at a low level throughout postmetamorphic life. In dark-reared larvae, synaptic ribbon density increases late in premetamorphic life and remains high into postmetamorphic life. The value for ribbon density in the adult animal maintained under room light (12-h light : 12-h darkness) is elevated above the oldest experimental light-reared larva. Dark-reared Stage 54 larvae have significantly higher ribbon density values than light-reared Stage 54 (P < 0.01). Dark-rearedMeta++ have significantly higher ribbon density than light-reared-Meta++ (P < 0.02). There are more ribbons at later stages in dark-reared larvae than at midlarval stages. The ribbon density value for R, is equivalent to that for lig!Tt-reared Stage 52. Rz, RR, and R-r have significantly more ribbons than light-reared larvae of the same stages, and these values are equivalent to those of dark-reared animals at the same stages. There were no significant differences at selected stages of development in the dimensions of ribbons in bipolar terminals of light- or dark-reared larvae (Table 5). Inner

Nuclear

Layer

Thickness. The average thickness of the inner nuclear layer does not change significantly with age or treatment through metamorphosis (Stage

144

TUCKER

Mean

Synaptic

IIcvelopmental stage

AND

HOLLYFIELD

TABLE

5

Ribbon

Size

(Micrometers)

Light-Reared

4.3 48 54 Meta++

Ilark-Reared

Bipolar

Receptor Cdl

Bipolar

Receptor Cell

0.1610 0.1580 0.1610 0.1980

0.4950 0.7620 0.7840 0.8990

0.1410 0.1490 0.1900 0.1690

0.6290 0.7240 0.7890 0.9130

54, light-reared : 2 = 25.3 pm; dark-reared: X = 28.1 pm; Stnge Mcfa++, light-reared : R = 28.9 pm ; dark-reared : r;l= 31.4 pm). Nftclear Density. The volume density of nuclei in the inner nuclear layer of light- and dark-reared larvae increases postmetamorphically (Stage 54, light-reared : 8 = 0.47/1000 pm3; dark-reared : 8 = 0.45/1000 q3; Stage Meta++, light-reared : 8 = 0.66/1000 pm3; dark-reared : fi = 0.83/1000 pm3) but this increase is not statistically significant. There is no significant difference at any stage in the mean size of nuclei in the inner nuclear layer of our light- and dark-reared larvae although they tend to be somewhat smaller in older animals (Stage 56, light-reared : 8 = 5.05 pm ; dark-reared : 8 = 7.86 pm ; Stage Meta++, light-reared : Z = 6.65 ,um; dark-reared : X = 6.48 pm). The number of layers of nuclei in the inner nuclear layer (3 to 4) did not change with age from Stage 45 through Meta++ in any specimen. In an earlier report, Hollyfield (17) found an increase from two to four layers of nuclei between Stage 55 and 65 (metamorphic climax). These TABLE Mean ’

Rod

Developmental stage 42 4.5 48 50 51 54 Meta+ Meta++

a P < 0.02.

Outer

Segment

6 Length

Light-reared

(Micrometers) Dark-reared _.

19.2 19.1 21.1 24.5 40.0 33.3 43.8 47.4

18.6 17.6 32.0 32.8~ 44.8 42.9a 36.6a 29.6a

INXER

PLESIFORM

SYNAPTIC

DENSITY

AND

LIGHT

145

observations are discussed collectively below in relation to changes with age and the effects of experimental treatment on other layers of the retina. Outer Retina Rod Oufu Segment Lengths. Measurements of rod cell outer segments were made from l-pm Epon sections taken parallel to the radius of the eye. These data reflect a near linear increase in outer segment length in both light- and dark-reared animals during larval stages and postn~etan~orl~l~ically as well in light-reared larvae. Outer segments are significantly longer in dark-reared larvae than in light-reared larvae premetamorphically at Stages 50 and 54 but decreased in length after metamorphosis (Table 6). Rccepfor Cell Ribbon Length. From Stage 41 through Meta++ there are no significant differences in synaptic ribbon lengths in photoreceptor bases in larvae under either rearing condition. The size of receptor cell ribbons tends to increase with age but this tendency is not statistically significant (Table 5). No attempt was made to distinguish between rod spherules and cone pedicles. DISCUSSION This study demonstrates differences in synaptic density in the inner plexiform layer of X. laevis retinae in response to development in cyclic light or darkness. The density of simple synapses in light-reared larvae is relatively high and stable until Stage 52. Thereafter, it decreases to adult levels. The same pattern is followed in dark-reared larvae but premetamorphically simple synapse density is significantly greater. We do not know whether amacrine to amacrine synapses comprise the bulk of those added in our dark-reared larvae. Certainly, in the rat (36)) this appears to be the case. Elevated density of simple synapses in dark-reared larvae is accompanied by a reduction in the expanse of the individual synapse profiles. However, at most stages the total amount of measured synaptic membrane per unit area of inner plexiform layer is the same in all specimens (Table 4). We were unable to detect a net change in the amount of synaptically specialized membrane in dark-reared larvae. Therefore, if new membrane is added in the dark to produce the larger observed volume densities of synapses, an equivalent amount of specialized membrane must be degraded. Whether or not this is the case, redistribution of synaptically specialized membrane seems to occur in dark-reared larvae such that lateral communication, probably between antacrine cells, is enhanced. The mean simple synapse density of dark-reared Meta++ (maintained in the dark for 11 months) was considerably higher than that of the companion light-reared Meta++ (darkreared : X = 343/1000 pm3 ; light-reared : .Z = 249/1000 pm3). This difference was not statistically significant as the confidence intervals overlap

146

TUCKER

AND

HOLLYFIELD

considerably. Only a limited number of mosaics was examined at this stage and this may have been a factor, In Rq (8 months dark-reared, 3 months recovery) the high simple synapse density (411/1000 &) with more restricted confidence limits was statistically different from its companion light-reared Meta++ value. This finding suggests that the density attained through Stage Meta+ in the dark was maintaiend but not modified by introduction to the light regime. The volume densities of bipolar cell ribbons are similar in dark- and light-reared larvae until Stage 52. During these early larval stages, values for dark-reared larvae are slightly higher than those for light-reared larvae, but this difference is not significant. By Stage 54 the former value is three times that of the latter. The elevated ribbon density in dark-reared larvae is maintained postmetamorphically and represents a shift away from the trend observed in light-reared animals where synaptic ribbon density decreases throughout larval life and continues to decrease following metamorphosis. Although the density of synaptic ribbon profiles is refractory to light deprivation during early larval stages, prolonged darkness does result in an increase in synaptic ribbon density. This finding is contrary to earlier findings in the rat (36)) where ribbon density in the inner plexiform layer was not modified by visual deprivation from application of opaque occluders. This discrepancy may be attributable to species differences or to the method and degree of deprivation in our respective studies. The synaptic ribbon density of our adult specimen was nearly three times higher than the density of ribbons in our oldest postmetamorphic lightreared larva. Although we do not know the photic history during development of this specimen, for the last 5 years it was maintained in our laboratory on a 12-h light : 12-hr dark cycle under bright room light ( > 500 lux) . It is unlikely that this longer term and more intense illumination would result in an elezuted ribbon density since Fifkova ( 10, 11) showed that, in rats, in response to excess light, ribbon density decreased. She noted that in adult rats the unsutured eye of a pair remains exposed to light for longer periods than eyes of untreated animals and that synaptic ribbon density was lower in the unsutured eye than in eyes of untreated animals. Adult Xenopus may exhibit a response in excess light which differs from that of rats due to species differences. Alternatively, since our adult specimen was at least 8 years old, the high ribbon density may be a change associated with aging. The simple synapse density for our first recovery animal (Rr, dark-reared to Stage 46, recovery Stages 46 to 52) was significantly lower than that in light-reared Stage 52 although it was not different from the simple synapse density in light-reared Stage 53. Introduction to the light regimen may then have accelerated development slightly in the inner plexiform layer of

INNER

PLEXIFORM

SYNAPTIC

DENSITY

AND

LIGHT

147

RI. It is also possible that RI was at late Stage 52 when fixed. We have the subjective impression that, although in most cases whole-body staging criteria are dependable, occasionally differentiation within specimens does not proceed synchronously and so there may be some morphological overlap between adjacent stages. Simple synapse density in Rz (dark-reared to Stage 51, recovery Stages 51 to 54) was midway between the values for dark- and light-reared larvae at Stage 54, indicating that this animal was in an early phase of recovery. However, the density of synaptic ribbons in Ra was not significantly different from the ribbon density of dark-reared larvae at stage 53. This suggests either that bipolar cell terminals are not responsive to the recovery regimen or that the return of synaptic ribbon density to light-reared values proceeds more slowly than the modulation of simple (primarily amacrine) synapses. Considering all the recovery data together, it is evident that a return of synaptic density to light-reared values is dependent on how young (by stage) the animal is when taken from the dark and placed under lightrearing conditions. RI was returned to the light at Stage 46, Rz at Stage 48, Rj at Stage 51, and Rd after metamorphic climax. The recovery data from these animals show a progressive decrease in the ability of ribbon and simple synaptic density values to return to light-reared levels. The failure of Kg to show significant differences in ribbon and simple synaptic densities from dark-reared larvae at the same stage after a loo-day exposure to a light : dark regime may be linked to changes in the physiology of the animal following metamorphosis. There was no difference in total growth rate between specimens under our rearing conditions. Our finding is in agreement with an earlier study (3) in which normal growth rate was maintained by dark-reared Amblysto~rla ~U~ZC~U~Z~IJL larvae. Recently, Besharse and Branclon (2) reported that, in an Ozark cave salamander, retinal degeneration following metamorphosis is more rapid when animals are maintained in the dark. We observed degenerative changes in the retinae of juvenile S. IntGs reared for nearlv 1 year in the dark. These included accumulation of masses of debris in the pigment epithelium, decreased rod outer segment length, and decreased thickness of the inner plexiform layer. These changes following prolonged darkness suggest that light may be required for maintenance of the structural integrity of the retina. This suggestion is supported by Ogneff (26) who reported that retinae of goldfish maintained 3 years in the dark lacked photoreceptors and Miiller cells and had degenerative changes in the inner plesiform layer and pigment epithelium. Nuclear density in the inner nuclear layer can provide critical information about intermediate neuronal input ratios between the photoreceptors and the ganglion cell layer. Data published by Fisher (12) correlate well

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with those we have presented here. Taken together, both observations suggest that in X. laevis there are only slight changes in nuclear density during normal larval development. Fisher (12) did not present data for inner nuclear layer nuclear size. In our dark-reared larvae there were more and smaller cells than in light-reared animals, yet these differences were not significant. Thus, responses of synapses in the inner plexiform layer to darkrearing cannot be linked at this time to changes in cell density. Several lines of evidence indicate that photoreceptor synaptic terminals continuously release a depolarizing transmitter substance in the dark and that the absorption of light hyperpolarizes the cell and causes a decrease in transmitter release (32-34, 38, 39, 45). Of the three types of neurons present in the inner plexiform layer, bipolar cells have direct connections with the photoreceptor terminal whereas processes of amacrine cells and ganglion cell dendrites are at least one synapse removed. In our dark-reared larvae, the increased density of synaptic ribbons in bipolar cell terminals and simple synapses in amacrine cell processes may represent a developmental response to the uninterrupted release of transmitter substance by the photoreceptor. Our data indicate that dark-rearing not only alters the density of simple synapses but, in the case of all conventional (largely amacrine) synapses, affects their size as well, though there is no significant increase in the amount of synaptically specialized membrane in dark-reared larvae to accommodate the elevated synaptic densities achieved. SUMMARY It is apparent that certain developmental changes occurring during retinal development in light- and dark-reared larvae can be linked in time to metamorphosis (Stages 55 to 65). In light-reared larvae the equilibrium between production and destruction of conventional synapses may be altered near metamorphosis, Simple conventional synaptic density levels decrease significantly in light-reared larvae just prior to early metamorphic stages and increase again to early larval levels several months postmetamorphically. There is no significant change in simple synapse density at any stage in dark-reared larvae. We found that in light-reared larvae ribbon density values decrease precipitously premetamorphically. Fisher (12) found that serial synaptic density remained relatively stable throughout larval life, increasing slightly near metamorphosis. We saw a slight insignificant increase in serial synaptic density postmetamorphically in all specimens. Ribbon synaptic density appears to become sensitive to light deprivation at late stages rather than at early larval stages, whereas simple conventional synaptic density is responsive to deprivation throughout development. We have shown that recovery to normal density values is possible only when the light regimen is initiated premetamorphically at either an early

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larval stage (ribbon density) or at midlarval stages (simple conventional synaptic density). Recovery does not occur when the light regimen is initiated postmetamorphically. Thus, normal retinal development, development in darkness, and recovery from the effects of dark-rearing show variability with respect to the onset and completion of metamorphosis. Permanence of the effects of dark-rearing is correlated with the duration of the deprivation period. We did not determine unequivocally whether there is a critical period during which sensitivity to dark-rearing is heightened (9, 14, 19, 22, 40). The maintenance of high simple synaptic density values in dark-reared larvae throughout Iarval life suggests that there is no such critical period in Xef1opzcs. Long-term dark-rearing not only affects the density of synapses but also results in decreased thickness of the inner plexiform layer, shortening of rod cell outer segments, and the accumulation of debris in the pigmented epithelium. Such changes are not seen in light-reared juveniles but are seen in aging adult retinae. Thus, retinal responses to dark-rearing may effectively mimic the effects of aging on the retina. As in other developing systems there appears to be an early overproduction of certain cellular components (in this case, conventional synapses and bipolar cell synaptic ribbons). Later these components become modified, their number stabilizing at adult levels. Depriving the animal of visual stimulation overrides this tendency to the mature state, thus emphasizing the influence of environment on the patterning of developmental processes. We cannot at this time define the manner by which light is operative in promoting patterned development of retinal synapses. We suggest that neurotransmitters released in the dark by photoreceptors may be implicated as causative agents in the elevation of synaptic density. REFERENCES 1. BARLOW, H. B. 1975. Visual experience 258 : 199-204. 2. BESHARSE, J. C., AND R. A. BRANDON.

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